Vaccinia virus presents a model system in which to study virus multiplication, pathogenesis, and regulatory mechanisms. The usefulness of a genetic approach to study virus replication has been established by temperature-sensitive (ts) mutants of bacteriophage and animal viruses. Because of the complexity of the vaccinia genome (over 100 genes) a successful genetic analysis will require a large number of mutants in different genes as determined by complementation experiments and biochemical characterization of their ts defects. In addition, the mutants must have low levels of reversion and leakiness. The primary aim of this research proposal is to isolate ts mutants of vaccinia virus and to determine their usefulness according to the above criteria. At the present time, 42 ts mutants that replicate at 33 degrees centigrade but not at 39.5 degrees centigrade have been isolated using a rapid screening procedure developed in this laboratory. These mutants are being characterized genetically by complementation and recombination experiments to identify the spectrum of genes represented; a qualitative complementation test has been developed to facilitate this analysis. The biochemical nature of their ts defects will be investigated by examining the synthesis of viral DNA, antigens, viral-specific polypeptides, and viral-induced enzymes at 39.5 degrees centigrade. In addition, the thermostability of infectivity and viral core enzymes will be investigated.